Amyloids are insoluble fibrous protein aggregates (also known as “amyloid fibrils” or “amyloid plaques”) which form in vivo, typically at hydrophobic-hydrophilic interfaces (HHIs), when naturally-occurring proteins and polypeptides misfold and inadvertently interact with one another and/or with other cell components to form aggregates. Amyloid plaques/fibrils, and consequencial amyloidosis, are believed to be responsible, at least in part, for numerous human diseases, including but not limited to Alzheimer's disease and type-2 diabetes.
Amyloid-beta (Aβ) peptides are the principle component of amyloid plaques found in the brains of Alzheimer's patients, and are thought to result from an amyloid precursor protein (APP) which when cut by particular enzymes yield Aβ peptides which misfold and aggregate to yield first oligomers and fibrils and ultimately amyloid plaques, amongst which sequence of conformers are those that are toxic to nerve cells.
Amylin (or Islet Amyloid Polypeptide—IAPP) is the principle component of amyloid deposits commonly found in pancreatic islets of type-2 diabetes patients. Such deposits of IAPP are thought to be typically initiated by unprocessed proIAPP seeds. It is thought that amylin, much like the related Aβ peptides associated with Alzheimer's disease, can induce apoptotic or excitotoxic cell-death in insulin-producing beta cells. It is thought that this effect may be relevant to the development of type 2 diabetes (Lorenzo A, Razzaboni B, Weir G C, Yankner B A (April 1994). “Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus”. Nature 368 (6473): 756-60).
Most proteins contain hydrophobic and hydrophilic domains and their adsorption to hydrophobic-hydrophilic interfaces (HHIs) is due to this amphiphilic character, conferring upon them surfactant properties. Protein adsorption, a complex process dependent on both protein and interface characteristics, is governed by bulk diffusion and results in interface adsorption. Once the interface becomes crowded, the adsorbed molecules will begin to align themselves at the interface (Wu et al 1993; Graham et al 1979; Ariola et al 2006). This conformation change may lead to intermolecular attractions between molecules, which in turn may result in the formation of interfacial multi-layers (Schmidt et al 1990). Protein adsorption also has significant implications in the activity of proteins toward membranes; amphiphilicity of proteins or peptides allow them to bind to membrane interfaces.
Amyloid peptides are amphiphilic and surface active (Lopes et al 2007; Cottingham M et al 2004; Soreghan et al 1994). This amphiphilicity has been exploited by amyloid precursor species to bind to membranes and to use this interaction to facilitate assembly into amyloid fibrils (Knight and Miranker 2004; Knight et al 2006; Lopes et al 2007; Terzi et al 1997). The eventual appearance of macroscopic amyloid deposits is a hallmark of protein misfolding diseases such as type II diabetes mellitus and Alzheimer's disease. During fibrillisation, amyloid peptides undergo a conformational change to form extended β-strands, facilitated by interaction with an HHI, whether membrane or AWI (air-water interface). This is due to a concentration effect of amphiphilic amyloids at the interface, which in turn alters the thermodynamic equilibrium and promotes peptide chains alignment (Lopes et al 2007; Terzi et al 1997; Soreghan et al 1994; Jean et al 2010). Recent attention has focused on determining the effects that lipids have on amyloid fibrillogenesis; in the presence of an AWI, anionic lipids were shown to enhance amyloid nucleation whereas zwitterionic lipids had no effect (Knight and Miranker 2004; Knight et al 2006; Chi et al 2008). However, this enhancing effect of anionic lipids on amyloidogenesis was demonstrated to be at its greatest in a context more closely mirroring in vivo conditions (the absence of an AWI; see Jean et al 2010). Thus, to understand fully fibrillisation enhancement by phospholipid bilayers, the biophysical consequences of amyloid surfactant activity and the potential importance of a competing AWI need to be taken into account.
Many proteins that adsorb at interfaces are able to form multi-layered proteinaceous networks, which can be stabilised by interfacial gel formation. The interfacial gel layer itself can be stabilised by numerous non-covalent interactions to form a meshwork of aggregates (e.g. fibres), typical of assembled amyloid polypeptides. To date gel formation has only been demonstrated for non-pathological amyloidogenic polypeptides (e.g. Escherichia coli curli, class II hydrophobins, b-lactoglobulin, spider silk) and for fragments of pathological amyloidogenic polypeptides (Wu et al 2012, Cox et al 2007, Bolisetty et al 2012, Yang et al 2012, Rijkers et al 2002, Krysmann et al 2008, Lepere et al 2007, Lakshmanan et al 2013, Manno M et al 2010). We have recently shown by direct rheological measurement that hydrated gel formation by pathological amyloid precursors including Aβ and islet amyloid precursor polypeptide (IAPP) does indeed occur at HHIs.
The pathologically relevant mechanisms of amyloid cytotoxicity are uncertain, although recent attention has focused on the concentration of amyloid peptides at membrane HHIs, which can cause disruption by a variety of mechanisms such as pore formation and membrane thinning (Kayed et al 2004; Quist et al 2005; Demuro et al 2005; Butterfield and Lashuel 2010). However, it seems probable that gel formation by amyloid species on the surface of cellular membranes could have important additional consequences for membrane integrity and cellular functions.
It is an object of the present invention to provide compounds that are capable of inhibiting the formation of amyloid deposits by acting at an early stage such that the production of the cytotoxic species, whether oligomer or fibril, is inhibited. Such compounds are potentially useful agents for the treatment of diseases such as Alzheimer's disease and/or type-2 diabetes.